PROJECT SUMMARY Large-scale bacteriophage genome sequencing projects are generating vast amounts of information, whereas the knowledge of how these diverse organisms function and what mechanisms are involved in phage-host interaction lags behind. In this project, we will focus on a phage genus from the contractile, long-tailed Myoviridae family, the Viunalikevirus. Specifically, we will investigate the host recognition mechanism of phage CBA120, known to infect the virulent Escherichia coli O157:H7. The Viunalikevirus genus, including CBA120, is unique because the genome of the phage encodes multiple putative tailspike proteins, exhibiting no significant amino acid sequence homology to one another except in the region that attaches to the phage baseplate. Typically, the non-contractile, short-tailed Podoviridae family members utilize tailspikes, whereas the majority of Myoviridae family members contain tail fibers. Unlike tail fibers, tailspikes enzymatically degrade the long lipopolysaccharides (O and K antigens) of environmental bacteria, which prevent the phage tail from reaching its high affinity receptor on the bacterial cell surface. Thus, the Viunalikevirus is expected to have unique receptor recognition mechanisms. Indeed, we have already discovered that one of the tailspike proteins of phage CBA120 is not involved in recognition of the O157 antigen, but instead is capable of degrading the exopolysaccharide of biofilms produced by diverse bacteria including both Gram-negative and Gram-positive species. We will investigate the CBA120 host range, receptor specificity, and enzymatic activity of each of four putative tailspikes to understand whether these proteins function in consort to recognize multiple receptors on the same host, or whether each tailspike recognizes another host. We will determine the crystal structures of each tailspike, identify potential active site residues, perform site-directed mutagenesis to probe the role of these residues, and obtain crystal structures of protein/ligand complexes to elucidate the structural basis for receptor recognition. The anti-biofilm properties of these tailspike proteins will also be evaluated using static and dynamic biofilms as well as extracted exopolysaccharides and LPS from sensitive organisms. In addition, we will characterize the stabilities and resistance to proteolysis of the full-length tailspike proteins and their truncated variants, to assess potential development of biologics against biofilm-forming bacteria that are resistant to standard care antibiotics. Results of the work will lay the foundation for producing new molecules that degrade biofilms and restore the efficacy of small molecule antibiotics.